An interesting group of proteins that has potentially many application possibilities is ice structuring proteins (ISP), often referred to as antifreeze proteins (AFP).
Warren et al. (U.S. Pat. No. 5,118,792) have suggested adding purified ISPs directly to food products prior to freezing to improve preservation characteristics during frozen storage. WO90/13571 A1 teaches methods of improving the freeze tolerance of food products by suppressing ice crystal growth or inhibiting ice recrystallization using antifreeze polypeptides in isolated form. A wide range of applications for ISP's in food and non-food have been suggested in the past, e.g. exemplified in Griffith and Vanya Ewart (1995) Biotechnology Advances 13:375-402 and EP2602263A2. However, many applications are currently economically not feasible due to the high cost in use of ISP.
Cost in use of such ISP's must be as low as possible to allow development of many different applications. Although the AFP type III HPLC12 from ocean pout is currently used for ice cream production on industrial scale, the difficulties associated with producing ISPs in large quantities at an economic attractive price preclude them from use in other industrial applications. An ideal ISP that can be used in different applications would be highly active at low concentration, low in cost, readily available, and simple to use.
A low cost in use of ISP can be obtained by selecting an ISP that has high ice structuring activity per mol protein. The minimal concentration of the best studied, and industrially used ISP (type III AFP from ocean pout) to obtain recrystallization inhibition in a 30% sucrose solution at −6° C. has been reported to be >700 nM (Smallwood et al (1999) Biochem. J. 340:385-391; Tomczak et al (2003) Biochem. BiophysRes. Comm. 311: 1041-1046). Consequently, a relatively high concentration of ISP is required in the application to obtain satisfactory results. Unilever has reported that the concentration of ISP (type III AFP) in ice cream application is ˜50 mg/kg (w/w) (Lewis (2006) Application for the Approval of Ice Structuring Protein Type III HPLC 12 Preparation for use in Edible Ices, Regulation (EC) No 258/97 of the European Parliament and of the Council of 27th January 1997 Concerning Novel Foods and Novel Food Ingredients), which is equivalent to 7 μM for this ISP. It has also been reported that ice crystal growth can be inhibited by 3-25 μM type III AFP (Li and Hew (1991) Protein engineering 4:1003-1008). An ISP which gives a similar effect with a lower required dosage would be beneficial for decreasing the cost-in-use of these proteins and would open up the possibility to develop additional applications.
Also high expression of ISP per kg fermentation broth will lead to a reduced cost price and a low cost-in-use. High expression will also lead to a more pure product that only requires a minimum of purification, thus further reducing cost price.
The productivity of ISP's that are currently described in literature is however low. Expression of type III AFP from ocean pout in the bakers' yeast Saccharomyces cerevisiae has been reported to be difficult (U.S. Pat. No. 6,914,043 B1) and only detectable when the culture broth supernatant was undiluted, suggesting a low level of expression.
Also expression of the ISP of Leucosporidium (LeIBP) in Escherichia coli or Pichia pastoris is described to be between 2.1 and 61.2 mg per liter culture broth in shake flask (Park et al (2012) Cryobiology 64:286-296), despite the track record of both microorganisms to successfully express heterologous proteins at high level.
Recently Lee et al have summarized all literature on the expression of known ISP's and concluded that expression levels do not exceed 175 mg/l (Lee et al (2013) Appl. Microbiol. Biotechnol. 97:3383-3393). They suggest that the application of ISP's is largely hampered by the lack of an economic production systems.
The same authors managed to increase productivity of LeIBP to ˜300 mg/l by a combined fed batch and induction of production by addition of methanol at reduced temperature. Due to the low productivity in fermentation, the protein had to be concentrated and purified before it could be used in further experiments, leading to a very poor yield and consequently a high cost price. Such measures may be useful for lab scale fermentation but are not economic for industrial production. Because of this the cost in use of the currently described ISP's is high and therefore the use of ISP in industry is limited.
Besides the cost in use it is also important that the end product has GRAS status (Generally Regarded As Safe) for the application of an ISP in a food product. Many of the ISPs described in literature are expressed in micro-organisms or produced with processes lacking this status and can therefore not be used in food applications. For example LeIBP is currently expressed in Pichia pastoris, a yeast which requires the addition of toxic methanol for induction of the expression of LeIBP (Lee et al (2013)).
Accordingly, there is a need for an ISP that is highly active, low in cost, readily available in food-grade form, and simple to use.